Polymer, contrast agent for nuclear magnetic resonance analysis or magnetic resonance imaging using the polymer, compound and method of nuclear magnetic resonance analysis and method of magnetic resonance imaging using the polymer

ABSTRACT

As a substance used as a contrast agent for a method of nuclear magnetic resonance analysis or a method of magnetic resonance imaging, a substance with high selectivity and high sensitivity was demanded. According to the present invention, when a polymer having, in a side chain thereof, a sequence of a  1 H— 13 C— 15 N,  1 H— 15 N— 13 C or  1 H— 13 C— 13 C bond, that is, a structure labeled with stable isotopes of  13 C and  15 N, is used, the abundance of such a sequence in one molecule can be increased, and hence, high selectivity and higher sensitivity can be attained when used as a contrast agent.

TECHNICAL FIELD

The present invention relates to a polymer, a contrast agent for nuclearmagnetic resonance analysis or magnetic resonance imaging using thepolymer, a compound, and a method of nuclear magnetic resonance analysisand a method of magnetic resonance imaging using the polymer.

BACKGROUND ART

Magnetic resonance imaging (hereinafter sometimes abbreviated as MRI),that is, an imaging method based on the principle of nuclear magneticresonance (NMR), is widely employed in medical settings because thismethod is only mildly invasive, has high spatial resolution and isexcellent for examining morphologies. In general, MRI images (notspectra) are obtained based on a difference in relaxation time and aregenerically used because of the problem of background noise caused bythe approximately 60% of water and lipid in a living body.

In general, ¹H NMR, ¹H is irradiated with pulses, so as to detect an NMRsignal therefrom. In contrast, in multiple resonance NMR, an NMR signalis detected by utilizing magnetization coherence transfer of the NMRsignal of ¹H to an adjacent NMR active nucleus, and by this method, aspecific chemical bond, such as a ¹H—¹³C, a ¹H—¹³C—¹⁵N sequence or a¹H—¹³C—¹³C sequence, can be selectively detected. Here, ¹³C and ¹⁵N arestable isotopes of ¹²C and ¹⁴N, respectively.

If a ¹H—¹³C sequence is to be selectively detected, the magnetizationtransfers from ¹H to ¹³C and then back to ¹H, and thus, the proton inthe ¹H—¹³C sequence can be detected. Alternatively, if a ¹H—¹³C—¹⁵Nsequence is to be selectively detected, the magnetization transfers from¹H through ¹³C to ¹⁵N and then back through ¹³C to ¹H, and thus, theproton in the ¹H—¹³C—¹⁵N sequence can be detected. This method islargely characterized by low natural abundances of these sequences. Forexample, the natural abundance of ¹H—¹³C—¹⁵N is as low as 0.0040%(because the natural abundances of ¹³C and ¹⁵N are 1.1% and 0.37%,respectively), and therefore, background noise that causes a problem inconventional magnetic resonance imaging is largely suppressed.Consequently, a compound containing a sequence of ¹H—¹³C—¹⁵N or the likeshows high selectivity and high sensitivity as a contrast agent fornuclear magnetic resonance analysis or magnetic resonance imaging.

PTL 1 discloses the following: A choline chloride labeled with ¹³C and¹⁵N is administered to a tumor-bearing mouse through a tail vein. Theliver, kidney and tumor extracted from the mouse 1 h after theadministration are ground, and the resultant mixture is subjected tocentrifugal separation for removing impurities. When the thus obtainedsupernatant solution is analyzed by ¹H—{¹³C—¹⁵N} triple resonance NMR, a¹H signal derived from a methyl group of choline at 3.0 ppm can bedetected.

CITATION LIST Patent Literature

PTL 1: Japanese Patent Application Laid-Open No. 2009-79046

SUMMARY OF THE INVENTION Technical Problem

The choline chloride labeled with ¹³C and ¹⁵N disclosed in PTL 1 hasnine ¹H—¹³C—¹⁵N sequences in one molecule. If the number of ¹H—¹³C—¹⁵Nsequences, ¹H—¹⁵N—¹³C sequences or ¹H—¹³C—¹³C sequences in one moleculeis further increased, the abundance of such sequences in a contrastagent can be increased, so that the nuclear magnetic resonance analysisand the magnetic resonance imaging can be conducted with higherselectivity and higher sensitivity.

The present invention was achieved in consideration of such a problem,and an object of the present invention is to provide a novel polymershowing high selectivity and high sensitivity in a method of nuclearmagnetic resonance analysis or a method of magnetic resonance imagingand a contrast agent including the polymer for use in nuclear magneticresonance analysis or magnetic resonance imaging.

Solution to the Problem

A polymer of the present invention is a polymer including, in a mainchain, one or a plurality of repeating unit(s) selected from the groupconsisting of the following Formulas (x1) to (x3) and having a degree ofpolymerization of two or more and 5000 or less, wherein a side chain ofeach of the repeating unit(s) has a structure selected from the groupconsisting of the following Formulas (y1) to (y3):

wherein in Formulas (x1) to (x3) above, each of R¹ to R⁴ independentlyrepresents a hydrogen atom, or a substituted or unsubstitutedhydrocarbon group having one or more and four or less carbon atoms; R⁵represents a substituted or unsubstituted hydrocarbon group having oneor more and nine or less carbon atoms, and if R⁵ is a hydrocarbon grouphaving two or more carbon atoms, any of the carbon atoms may be bound tothe side chain, in Formulas (x1) to (x3) above, the symbol * (anasterisk) represents a bond to the side chain directly or via a linker,in Formulas (y1) to (y3) above, the symbol * (an asterisk) represents abond to the main chain directly or via a linker, in Formulas (y1) to(y3) above, Z represents an arbitrary monovalent atom or monovalent atomgroup, in Formula (y3) above, R⁶ represents a direct bond, or asubstituted or unsubstituted hydrocarbon group having one or more andfour or less carbon atoms, and a substituent of each of R¹ to R⁶ is afunctional group including at least one selected from the groupconsisting of a halogen atom, an oxygen atom and a nitrogen atom.

A compound of the present invention is represented by any one of thefollowing Formulas (j1) to (j12)

wherein, in Formulas (j1) to (j12) above, each of a and b independentlyrepresents an integer of one or more and four or less, and a hydrogenatom of a methylene group is optionally replaced by another atom.

A method of nuclear magnetic resonance analysis of the presentinvention, including detecting a polymer in a specimen, includes:preparing the polymer; providing the polymer to the specimen; andapplying electromagnetic waves to the specimen provided with thepolymer, wherein magnetization transfer (coherence transfer) amongnuclei in a ¹H—¹³C—¹⁵N bond sequence, a ¹H—¹⁵N¹³C bond sequence or a¹H—¹³C—¹³C bond sequence of the polymer is utilized for detecting thepolymer.

A method of magnetic resonance imaging of the present invention,including detecting the position of a polymer in a specimen, includes:preparing the polymer; providing the polymer to the specimen; andapplying electromagnetic waves to the specimen provided with thepolymer, wherein magnetization transfer (coherence transfer) amongnuclei in a ¹H—¹³C—¹⁵N bond sequence, a ¹H—¹⁵N—¹³C bond sequence or a¹H—¹³C—¹³C bond sequence of the polymer is utilized for detecting theposition of the polymer.

Advantageous Effects of the Invention

Using a polymer of the present invention, because the polymer includes,in side chains thereof, a large number of sequences of ¹H—¹³C—¹⁵N,¹H—¹⁵N—¹³C or ¹H—¹³C—¹³C, that is, a structure labeled with the stableisotopes ¹³C and ¹⁵N, the abundance of such a sequence in one moleculecan be increased, so that high selectivity and higher sensitivity can beattained.

Further features of the present invention will become apparent from thefollowing description of exemplary embodiments with reference to theattached drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a ¹H NMR spectrum of 2-((2-cyanoethoxy)(diisopropylamino)phosphinooxy)ethyl methacrylate (Formula (m6)).

FIG. 2 is a ¹H NMR spectrum of a ¹³C/¹⁵N labeled MPC (of Formula (j1)wherein a=2 and b=2).

FIG. 3 is a ¹³C NMR spectrum of the ¹³C/¹⁵N labeled MPC (of Formula (j1)wherein a=2 and b=2).

FIG. 4 is a ¹⁵N NMR spectrum of the ¹³C/¹⁵N labeled MPC (of Formula (j1)wherein a=2 and b=2).

FIG. 5 is a ³¹P NMR spectrum of the ¹³C/¹⁵N labeled MPC (of Formula (j1)wherein a=2 and b=2).

FIG. 6 is a ¹H NMR spectrum of a ¹³C/¹⁵N labeled PMPC p2-3 (of Formula(p2) wherein n=40, and M_(n) (GPC)=12000).

FIG. 7 is a ¹³C NMR spectrum of the ¹³C/¹⁵N labeled PMPC p2-3 (ofFormula (p2) wherein n=40, and M_(n) (GPC)=12000).

FIG. 8 is a ¹⁵N NMR spectrum of the ¹³C/¹⁵N labeled PMPC p2-3 (ofFormula (p2) wherein n=40, and M_(n) (GPC)=12000).

FIG. 9 is a GPC profile of a ¹³C/¹⁵N labeled PMPC p2-1 (of Formula (p2)wherein n=23, and M_(n) (GPC)=6800)

FIG. 10 is a GPC profile of a ¹³C/¹⁵N labeled PMPC p2-2 (of Formula (p2)wherein n=16, and M_(n) (GPC)=5000).

FIG. 11 is a GPC profile of a ¹³C/¹⁵N labeled PMPC p2-3 (of Formula (p2)wherein n=40, and M_(n) (GPC)=12000)

FIG. 12 is a pulse sequence for ¹H—{¹³C—¹⁵N} triple resonance NMR.

FIG. 13A is a ¹H NMR spectrum of the ¹³C/¹⁵N labeled PMPC p2-3 in amouse liver extract.

FIG. 13B is a ¹H—{¹³C} double resonance NMR spectrum of the ¹³C/¹⁵Nlabeled PMPC p2-3 in a mouse liver extract.

FIG. 13C is a ¹H—{¹³C—¹⁵N} triple resonance NMR spectrum of the ¹³C/¹⁵Nlabeled PMPC p2-3 in a mouse liver extract.

FIGS. 14A and 14B show a macromolecular effect (effect of accumulationof stable isotopes) on ¹H—{¹³C—¹⁵N} triple resonance signal sensitivity.

FIG. 15A is a ¹H NMR spectrum of a ¹³C/¹⁵N labeled PMPC p4-scFvconjugate.

FIG. 15B is a ¹H—{¹³C} double resonance NMR spectrum of the ¹³C/¹⁵Nlabeled PMPC p4-scFv conjugate.

FIG. 15C is a ¹H—{¹³C—¹⁵N} triple resonance NMR spectrum of the ¹³C/¹⁵Nlabeled PMPC p4-scFv conjugate.

DESCRIPTION OF EMBODIMENT

A contrast agent according to the present invention includes a polymer,and the polymer has, in side chains thereof, one or a plurality ofsequences selected from the group consisting of a ¹H—¹³C—¹⁵N sequence, a¹H—¹⁵N—¹³C sequence and a ¹H—¹³C—¹³C sequence that are detectable by theprinciple of nuclear magnetic resonance. These sequences are detectedbased on similar principles, and for example, a ¹H—¹³C—¹⁵N sequence canbe detected by transferring the magnetization of ¹H to ¹³C, transferringthe magnetization of ¹³C to ¹⁵N, returning the magnetization of ¹⁵N to¹³C, and returning the magnetization of ¹³C to ¹H. Similarly, a¹H—¹⁵N—¹³C sequence can be detected by transferring the magnetization of¹H to ¹⁵N, transferring the magnetization of ¹⁵N to ¹³C, returning themagnetization of ¹³C to ¹⁵N and returning the magnetization of ¹⁵N to¹H. In addition, a ¹H—¹³C—¹³C sequence can be similarly detected. Thecontrast agent including the polymer having such a structure permitsnuclear magnetic resonance analysis and magnetic resonance imaging withhigher selectivity and higher sensitivity because a large number of suchsequences detectable by the principle of nuclear magnetic resonance areincluded in the side chains. It is noted that a plurality of the abovesequences can be included per side chain unit.

Because ¹⁵N and ¹³C used in these sequences are stable isotopes, thereis no danger of exposure and no restriction in handling time as comparedwith conventional labeled compounds using radioactive isotopes.

An embodiment of the present invention will now be described; however,the present invention is not limited to the embodiment.

(Polymer)

A side chain of a polymer of the present embodiment has a structureselected from the group consisting of the following Formulas (y1) to(y3).

In Formulas (y1) to (y3) above, the symbol * (an asterisk) represents abond to a main chain directly or via a linker. In Formulas (y1) to (y3)above, Z represents an arbitrary monovalent atom or monovalent atomgroup.

In Formula (y3) above, R⁶ represents a direct bond, or a substituted orunsubstituted hydrocarbon group having one or more and four or lesscarbon atoms. A substituent of R⁶ is a functional group including atleast one selected from the group consisting of a halogen atom, anoxygen atom and a nitrogen atom.

As described above, because a side chain of the polymer of the presentembodiment includes at least one of the structures of Formulas (y1) to(y3), namely, the sequence of ¹H—¹³C—¹⁵N, ¹H—¹⁵N—¹³C or ¹H—¹³C—¹³C, thepolymer can be used as a contrast agent with high selectivity in themethod of nuclear magnetic resonance analysis or the method of magneticresonance imaging. Furthermore, because many of such sequences areincluded, the polymer can be used as a highly sensitive contrast agentin the method of nuclear magnetic resonance analysis or the method ofmagnetic resonance imaging. The polymer may include one or more of thesesequences and preferably includes at least the ¹H—¹³C—¹⁵N sequence.

In the polymer of the present embodiment, the linker and the side chaincan be selected from the group consisting of the following Formulas (y5)to (y7).

In Formulas (y5) to (y7) above, the symbol * (an asterisk) represents abond to the main chain. In Formulas (y5) to (y7) above, each of a and bindependently represents an integer of one or more and four or less, anda hydrogen atom of a methylene group of the above formulas is optionallyreplaced by another atom.

If a structure similar to choline is included as in Formula (y5) above,the polymer of the present embodiment is evidence of high tumoraccumulation. Alternatively, if a carboxyl group is included as inFormula (y6) above, the structure is preferable because the carboxylgroup can be easily bound to a trapping molecule that specifically bindsto a target site such as an antibody. Alternatively, if a sulfonic groupis included as in Formula (y7) above, the structure is preferablebecause the structure is highly hydrophilic and minimally aggregates inan organism.

(Main Chain)

The polymer of the present embodiment can include, as a main chain, arepeating unit represented by any one of the following Formulas (x1) to(x3).

In Formulas (x1) to (x3) above, each of R¹ to R⁴ independentlyrepresents a hydrogen atom, or a substituted or unsubstitutedhydrocarbon group having one or more and four or less carbon atoms, andR⁵ represents a substituted or unsubstituted hydrocarbon group havingone or more and nine or less carbon atoms. If R⁵ is a hydrocarbon grouphaving two or more carbon atoms, any of the carbon atoms may be bound tothe side chain. A substituent of each of R¹ to R⁵ is a functional groupincluding at least one selected from the group consisting of a halogenatom, an oxygen atom and a nitrogen atom.

In Formulas (x1) to (x3) above, the symbol * (an asterisk) represents abond to the side chain directly or via a linker.

The polymer of the present embodiment has a degree of polymerization ofpreferably two or more and 5000 or less. In addition, the degree ofpolymerization of the polymer of the present embodiment is preferably 10or more and more preferably 20 or more. If the degree of polymerizationis 10 or more, the sensitivity is high, and if the degree ofpolymerization is 20 or more, the sensitivity is even higher.

The degree of polymerization of the polymer of the present embodiment ispreferably 1000 or less and more preferably 400 or less. If the degreeof polymerization is 1000 or less, the viscosity is low, which ispreferable in administering the polymer of the present embodiment to anorganism. The degree of polymerization of the polymer of the presentembodiment is preferably 10 or more and 1000 or less, more preferably 10or more and 400 or less, and still more preferably 20 or more and 400 orless.

The main chain may be in the form of a straight chain or may have abranched structure.

The repeating unit of the main chain may include one repeating unit ortwo or more repeating units.

(Exemplary Embodiment of Polymer)

In the polymer of the present embodiment, the repeating unit of the mainchain can include merely the repeating units of the above Formulas (x1)to (x3). In this case, a structure including the repeating unit isrepresented by the following Formula (I).

In Formula (I) above, X represents any one of the following Formulas(x1) to (x3), and the polymer has a degree of polymerization of two ormore and 5000 or less. In the following Formulas (x1) to (x3), thesymbol * (an asterisk) represents a bond to L of the above Formula (I)or a bond to Y when L represents a direct bond. In the followingFormulas (x1) to (x3), each of R¹ to R⁴ independently represents ahydrogen atom, or a substituted or unsubstituted hydrocarbon grouphaving one or more and four or less carbon atoms, and R⁵ represents asubstituted or unsubstituted hydrocarbon group having one or more andnine or less carbon atoms. A substituent of R¹ to R⁵ is a functionalgroup including at least one selected from the group consisting of ahalogen atom, an oxygen atom and a nitrogen atom.

In Formula (I) above, L is a direct bond or an arbitrary divalent atomor divalent atom group. If L is an arbitrary divalent atom or divalentatom group, L binds to X or Y of Formula (I) above.

In Formula (I) above, Y is represented by any one of Formulas (y1) to(y3) below. In the following Formulas (y1) to (y3), the symbol * (anasterisk) represents a bond to L of Formula (I) above or a bond to X ifL is a direct bond, and Z represents an arbitrary monovalent atom ormonovalent atom group. In the following Formula (y3), R⁶ represents adirect bond, or a substituted or unsubstituted hydrocarbon group havingone or more and four or less carbon atoms. A substituent of R⁶ is afunctional group including at least one selected from the groupconsisting of a halogen atom, an oxygen atom and a nitrogen atom.

Furthermore, in the polymer of the present embodiment, L of Formula (I)can be selected from the group consisting of a substituted orunsubstituted hydrocarbon group having one or more and four or lesscarbon atoms and the following Formulas (l1) to (l3).

A substituent of the hydrocarbon group is a functional group includingat least one selected from the group consisting of a halogen atom, anoxygen atom and a nitrogen atom.

In Formulas (l1) to (l3) above, the symbol * (an asterisk) represents abond to X or Y of Formula (I).

In Formulas (l1) to (l3) above, L′ is selected from the group consistingof a substituted or unsubstituted hydrocarbon group having one or moreand four or less carbon atoms and the following Formula (l′).

In Formula (l′) above, each of a and b independently represents aninteger of one or more and four or less, and the hydrocarbon grouphaving one or more and four or less carbon atoms of L′ and a hydrogenatom of a methylene group of Formula (l′) is optionally replaced byanother atom such as a halogen atom, an oxygen atom or a nitrogen atom.

Furthermore, in the polymer of the present embodiment, Z of Formula (y1)or (y2) can be represented by the following Formula (z1), or Z ofFormulas (y1) to (y3) can be represented by the following Formula (z2)because high sensitivity may be thus attained.

*—¹⁵NH₂  (z1)

*—¹³CH₃  (z2)

In Formulas (z1) and (z2) above, the symbol * (an asterisk) represents abond to ¹³C or ¹⁵N of Formulas (y1) to (y3).

In the polymer of the present embodiment, if Z of Formulas (y1) to (y3)is represented by Formula (z3) or (z4) below, the polymer is preferredbecause the polymer can be easily bound to an antibody and is highlyhydrophilic.

*—(CH₂)_(d)—COO⁻  (z3)

*—(CH₂)_(d)—SO₃ ⁻  (z4)

In Formulas (z3) and (z4) above, the symbol * (an asterisk) represents abond to ¹³C or ¹⁵N of Formulas (y1) to (y3). In Formulas (z3) and (z4)above, d represents an integer of one or more and four or less, and ahydrogen atom of a methylene group is optionally replaced by anotheratom such as a halogen atom, an oxygen atom or a nitrogen atom.

If the above Formula (z2) forms a structure similar to choline, thepolymer of the present embodiment is able to show high tumoraccumulation. If a carboxyl group is included as in Formula (z3) above,the structure is preferable because the carboxyl group can be easilybound to a trapping molecule that specifically binds to a target sitesuch as an antibody. Alternatively, if a sulfonic group is included asin Formula (z4) above, the structure is preferable because the polymeris highly hydrophilic and minimally aggregates in an organism.

Examples of the polymer of the present embodiment include the followingFormulas (i1) to (i12).

In Formulas (l1) to (i12) above, each of a and b independentlyrepresents an integer of one or more and four or less, and a hydrogenatom of a methylene group is optionally replaced by another atom.

Incidentally, the polymer of the present embodiment is hereinaftersometimes designated as the ¹³C/¹⁵N labeled polymer (¹³C/¹⁵N-PMPC).

(Trapping Molecule)

The polymer of the present embodiment can include a trapping moleculethat specifically binds to a target site. Examples of a trappingmolecule that specifically binds to a target site include a substancethat specifically binds to a target site of a tumor or the like, and asubstance that specifically binds to a substance present around a targetsite, and the trapping molecule may be selected freely from chemicalsubstances including biomolecules and medicines. Specific examples ofthe trapping molecule include antibodies, antibody fragments, enzymes,biologically active peptides, glycopeptides, sugar chains, lipids,nucleic acids and molecular recognition compounds. One of thesesubstances may be used singly, or a plurality of the substances may beused in combination. When the polymer of the present embodimentincluding a chemically bound trapping molecule is used, a specifictarget site can be detected, or dynamics, localization, drug effects,metabolism or the like of a target substance can be traced.

Here, an antibody is a general term for proteins belonging to theimmunoglobulin family induced by an immune system in response to aspecific molecule (antigen), and has the property of binding to theantigen. An example of the immunoglobulin family includes immunoglobulinG (hereinafter sometimes abbreviated as IgG). Here, the antibody may bea polyclonal antibody or a monoclonal antibody. Incidentally, anantibody portion may include, apart from an antibody, any amino acid aslong as the antigen-binding capacity is not harmed.

The antibody is not limited to a whole antibody but may be an antibodyfragment. An antibody fragment means a derivative of an antibodyobtained by reducing the molecular weight with the property of bindingto a specific molecule retained. Examples of an antibody fragmentinclude a Fab fragment, a Fab′ fragment, a F(ab′)2, polypeptideincluding a variable heavy chain (VH) domain alone, a variable lightchain (VL) domain alone, a composite of VH and VL, a camelized VH domainor a complementary determining region (CDR) of an antibody, and a singlechain antibody (single chain Fv, hereinafter sometimes abbreviated asscFv) that is a polypeptide including a VH region and a VL region of anantibody linked with peptide linker. Because an antibody fragment has asmaller molecular size than a whole antibody, an antibody fragment hashigh tissue permeability and a high clearance rate, and hence issuitable for use in a diagnostic agent or a contrast agent.

Of antibody fragments, a single chain antibody is preferred. This isbecause a single chain antibody can be inexpensively and easily preparedcorresponding to each of various antigens, and has a smaller molecularweight than whole antibodies and antibody fragments other than thesingle chain antibody, and hence is rapidly removed from a body oreasily reaches a lesion site. Consequently, a single chain antibody issuitable for use in detecting or treating a lesion site.

A method for binding a trapping molecule to the polymer of the presentembodiment depends upon the type of trapping molecule to be used, butany of the known methods can be employed. For example, a method in whicha reaction is caused between a functional group at an end of the polymerof the present embodiment and a functional group of the trappingmolecule for attaining a chemical bond can be employed.

If the functional group at an end of the polymer of the presentembodiment is a maleimide group, a target substance can be obtained bycausing a reaction with a trapping molecule having a thiol group.Alternatively, if the functional group at an end of the polymer of thepresent embodiment is an N-hydroxysuccinimide group, a target substancecan be obtained by causing a reaction with a trapping molecule having anamino group. After the reaction, the polymer having the trappingmolecule bound thereto can be separated and purified.

(Copolymer)

The polymer of the present embodiment may be a copolymer having two ormore repeating units. If the polymer of the present embodiment is acopolymer, the copolymer may be any one of an alternating copolymer, arandom copolymer and a block copolymer.

If the polymer of the present embodiment is a copolymer, the copolymermay include, apart from the aforementioned repeating unit(s) included inthe main chain, a repeating unit derived from a methacrylate monomer, arepeating unit derived from a methacrylamide monomer, a repeating unitderived from an amino acid monomer or a repeating unit derived from ahydroxy acid monomer.

If the polymer of the present embodiment is a copolymer, the copolymermay include, apart from the aforementioned repeating unit(s) included inthe main chain, a repeating unit not having the side chain of thepresent embodiment but represented by any one of the following Formulas(a1) to (a3).

In Formulas (a1) to (a3) above, R⁷ represents an arbitrary monovalentatom or monovalent atom group and is a hydrogen atom or preferably asubstituted or unsubstituted hydrocarbon group having one or more andsix or less carbon atoms. A substituent of R⁷ is a functional groupincluding at least one selected from the group consisting of a halogenatom, an oxygen atom and a nitrogen atom.

Incidentally, in the case where the polymer of the present embodiment isa copolymer, all repeating units included in the copolymer may have, inside chains thereof, the structures represented by Formulas (y1) to (y3)described above, or merely some of the repeating units included in thecopolymer may have the structures represented by Formulas (y1) to (y3)described above. In the former case, extremely high sensitivity can beattained when the copolymer is used as a contrast agent for the multipleresonance NMR. On the other hand, in the latter case, because arepeating unit having none of the structures represented by the aboveFormulas (y1) to (y3) is used, the copolymer can be designed accordingto a purpose; for example, to modify the hydrophilic property orabsorption ability in an organism.

(Polymer End)

The end of the repeating unit of the polymer of the present embodimentis not especially limited but preferably includes any one of anN-hydroxysuccinimide group, a maleimide group, an amino group, an azidegroup, an ethynyl group, a vinyl group, a trichlorosilyl group, a thiolgroup, a hydroxyl group and an alkyl group. Such a functional group canbe easily bound to a trapping molecule that specifically binds to atarget site such as an antibody. The end of the polymer of the presentembodiment has, for example, any one of the structures of the followingFormulas (b1) to (b9).

In Formulas (b1) to (b9) above, e represents an integer of one or moreand 11 or less, f represents an integer of zero or more and 17 or less,and R⁸ represents a hydrogen atom or a methyl group. Both ends of thepolymer of the present embodiment may have the same structure ordifferent structures. In Formulas (b1) to (b9) above, the symbol * (anasterisk) represents a bond to a repeating unit of the main chain.

(Contrast Agent for Nuclear Magnetic Resonance Analysis or MagneticResonance Imaging)

A contrast agent for nuclear magnetic resonance analysis or magneticresonance imaging of the present embodiment includes the aforementionedpolymer and a dispersion medium. Here, a dispersion medium is a liquidsubstance for dissolving the above polymer therein, and examples includea physiological saline solution, distilled water for injection and aphosphate buffer solution (PBS). Furthermore, in addition to thedispersion medium, the contrast agent may include a pharmacologicallyacceptable additive if necessary. In the contrast agent for the nuclearmagnetic resonance analysis or the magnetic resonance imaging of thepresent embodiment, the above polymer may be previously dissolved in thedispersion medium, or the above polymer and the dispersion medium may beprovided as a kit so that the polymer can be dissolved in the dispersionmedium before administration to an organism. The contrast agent of thepresent embodiment can be accumulated in a larger amount in a tumor sitethan in a normal region within an organism owing to the enhancedpermeability and retention (EPR) effect. By detecting the accumulatedcontrast agent by the method of nuclear magnetic resonance analysis orthe method of magnetic resonance imaging, the presence of a tumor can bedetected or a specific tumor site can be imaged.

(Compound)

The polymer of the present embodiment can be synthesized by polymerizinga polymerizable compound by a known method. The polymerizable compoundmay have the structures represented by Formulas (y1) to (y3) mentionedabove, or alternatively, the structures represented by the aboveFormulas (y1) to (y3) may be added to the polymer by modifying a sidechain after the polymerization. For example, the polymer can besynthesized by polymerizing compounds of the following Formulas (j1) to(j12).

In Formulas (j1) to (j12) above, each of a and b independentlyrepresents an integer of one or more and four or less, and a hydrogenatom of a methylene group is optionally replaced by another atom such asa halogen atom, an oxygen atom or a nitrogen atom. Incidentally, thecompound of the present embodiment is hereinafter sometimes designatedas the ¹³C/¹⁵N labeled monomer (¹³C/¹⁵N-MPC).

The polymer of the present embodiment mentioned above can be prepared bypolymerizing these compounds.

(Polymerization of the Compound)

For the polymerization of the compound of the present embodiment, any ofthe conventionally known polymerization methods can be appropriatelyselected according to the type of compound.

If, for example, a vinyl monomer is selected as the above polymerizablecompound, the living radical polymerization method, particularly theatom transfer radical polymerization (ATRP) method, may be employed asthe polymerization method. Because the ATRP method is simple and themolecular weight can be easily controlled, the ATRP method is preferred.

If hydroxy acid or amino acid is selected as the polymerizable compound,condensation polymerization may be employed as the polymerizationmethod. In employing condensation polymerization, a condensation agentmay be appropriately used.

If lactide, lactone or lactam is selected as the polymerizable compound,ring-opening polymerization may be employed as the polymerizationmethod. In employing the ring-opening polymerization, a catalyst may beappropriately used.

In the polymerization of the polymerizable compound, two or morecompounds having different structures may be used for producing analternating copolymer, a random copolymer or a block copolymer.

(Atom Transfer Radical Polymerization)

In the above ATRP method, a polymerization initiator having a highlyreactive carbon-halogen bond and a transition metal complex working as apolymerization catalyst are used to polymerize a vinyl monomer. The¹³C/¹⁵N labeled polymer (¹³C/¹⁵N-PMPC) of the present embodiment can beobtained, for example, by the atom transfer radical polymerization asshown in the following Reaction Formula 1.

In the present embodiment, instead of producing a homopolymer, analternating copolymer, a random copolymer or a block copolymer can beproduced by using two or more vinyl monomers having differentstructures.

If a vinyl monomer is polymerized by the atom transfer radicalpolymerization, a polymerization initiator—for example, represented byany one of Formulas (k1) to (k9) below can be used. In the followingFormulas (k1) to (k9), e represents an integer of 1 or more and 11 orless, f represents an integer of 0 or more and 17 or less, and R⁸represents a hydrogen atom or a methyl group.

Under an inert gas atmosphere, a polymerization initiator and atransition metal complex are added to a reaction medium inducing a vinylmonomer, so as to conduct the atom transfer radical polymerization. Thepolymerization proceeds in a living manner, so that a polymer with asmall molecular weight distribution can be obtained.

The reaction medium is not especially limited, but, for example, water,methanol, ethanol, dimethyl sulfoxide, dimethyl formamide andacetonitrile may be used. One such medium may be used singly or two ormore of the media may be used in combination. As the inert gas, nitrogengas or argon gas may be used.

The transition metal complex to be used includes a halogenated metal anda ligand. As metal species of the halogenated metal, transition metalsfrom Ti with atomic number 22 to Zn with atomic number 30 are preferred,and Fe, Co, Ni and Cu are particularly preferred. Of these halogenatedmetals, cuprous chloride and cuprous bromide are preferred.

The ligand is not especially limited as long as the ligand can becoordinated in a halogenated metal, and for example, 2,2′-bipyridyl,tris(2-dimethylaminoethyl)amine, ethylenediamine, dimethylglyoxime,1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane,1,10-phenanthroline, N,N,N′,N″,N″-pentamethyldiethylenetriamine,tris(2-aminoethyl)amine or the like can be used.

The polymerization temperature is in the range of 0° C. to 80° C., andpreferably in the range of 10° C. to 60° C.

(Synthesis of Compound)

The compounds represented by Formulas (j1) to (j12) above can besynthesized by known methods. For example, the compound represented bythe above Formula (j1) (wherein a=2 and b=2) can be synthesizedaccording to Reaction Formula 2 below. To introduce a ¹³C nucleus and a¹⁵N nucleus into 2-methacryloyloxyethyl phosphorylcholine (MPC), ¹⁵Nglycine represented by Formula (m1) is used as a starting material, and¹³C methyl iodide is used to introduce the ¹³C nucleus. In this way, thetargeted vinyl monomer (¹³C/¹⁵N-MPC) represented by Formula (j1) can beobtained in a yield of 48% (overall).

(Contrast Agent)

When the atom transfer radical polymerization is conducted using apolymerization initiator represented by any of Formulas (k1) to (k9)above and a ¹³C/¹⁵N labeled monomer, a polymer having a side chaindouble-labeled with stable isotopes of a ¹³C nucleus and a ¹⁵N nucleuscan be obtained.

Onto the polymer obtained by the atom transfer radical polymerizationusing the polymerization initiator represented by any of Formulas (k1)to (k9) and the ¹³C/¹⁵N labeled monomer, a trapping molecule thatspecifically binds to a target site can be immobilized. However, in thecase of Formula (k1), deprotection needs to be conducted for changinginto a maleimide group.

(Method of Nuclear Magnetic Resonance Analysis)

A method of nuclear magnetic resonance analysis of the presentembodiment includes detecting a polymer included in a specimen andfurther includes: preparing a ¹³C/¹⁵N labeled polymer corresponding tothe polymer; providing the polymer to the specimen; and applyingelectromagnetic waves to the specimen provided with the polymer; andmagnetization transfer (coherence transfer) among nuclei in a ¹H—¹³C—¹⁵Nbond sequence, a ¹H—¹⁵N—¹³C bond sequence or a ¹H—¹³C—¹³C bond sequenceof the polymer is utilized for detecting the polymer. In the presentembodiment, any one or more of these sequences may be used for detectingthe polymer, and at least the ¹H—¹³C—¹⁵N sequence can be used.

(Method of Magnetic Resonance Imaging)

A method of magnetic resonance imaging of the present embodimentincludes detecting the position of a polymer in a specimen and furtherincludes: preparing a ¹³C/¹⁵N labeled polymer corresponding to thepolymer; providing the polymer to the specimen; and applyingelectromagnetic waves to the specimen provided with the polymer; andmagnetization transfer (coherence transfer) among nuclei in a ¹H—¹³C—¹⁵Nbond sequence, a ¹H—¹⁵N—¹³C bond sequence or a ¹H—¹³C—¹³C bond sequenceof the polymer is utilized for detecting the position of the polymer. Inthe present embodiment, any one or more of these sequences may be usedfor detecting the polymer, and at least the ¹H—¹³C—¹⁵N sequence can beused.

When a contrast agent in which a trapping molecule is bound is used inthe method of magnetic resonance imaging, the position of a tumor or thelike can be detected through the aforementioned steps.

In providing the polymer as described above, the method foradministering the polymer of the present embodiment to an organism isnot especially limited, and a method of injection, oral administrationor the like can be employed.

Furthermore, in the case where the polymer is used in an organism,various specific target sites can be detected by appropriately selectingthe trapping molecule. If, for example, a substance that binds to aspecific tumor is used as the trapping molecule, the specific tumor canbe detected. Alternatively, if a substance that binds to a specificliving substance such as a protein or an enzyme present in a largeamount around a specific disease site is used as the trapping molecule,the specific disease can be detected. Furthermore, the polymer of thepresent embodiment can be used for detecting a tumor owing to the EPReffect, even when the trapping molecule is not included.

EXAMPLES

Specific reagents, reaction conditions and the like employed inpreparing a polymer of the present invention in each example will now bedescribed, but the reagents, reaction conditions and the like can bechanged or modified, and these changes and modifications are connoted inthe present invention. Consequently, the examples described below aregiven for the purpose of assisting in the understanding of the presentinvention and are not intended to limit the scope of the presentinvention in any way.

(Method for Measuring the NMR Spectrum)

The ¹H NMR spectrum was measured using a JEOL EX400 (400 MHz,manufactured by JEOL Ltd.) or a JEOL AL300 (300 MHz, manufactured byJEOL Ltd.).

The ¹³C NMR spectrum was measured using a JEOL EX400 (100 MHz,manufactured by JEOL Ltd.) or a JEOL AL300 (75 MHz, manufactured by JEOLLtd.).

The ¹⁵N NMR spectrum was measured using an ECX400P (40 MHz, manufacturedby JEOL Ltd.). The ³¹P NMR spectrum was measured using an ECX400P (160MHz, manufactured by JEOL Ltd.).

(Method for Measuring the Molecular Weight)

The electrospray ionization time-of-flight mass (ESI-TOF MS) spectrumwas measured using a micrOTOF focus-KE spectrometer (manufactured byBruker Daltonics).

The molecular weight was measured by gel permeation chromatography (GPC)(ChromNAV, manufactured by JASCO Corporation).

(Method for Measuring Multiple Resonance NMR)

The ¹H—{¹³C} double resonance NMR and the ¹H—{¹³C—¹⁵N} triple resonanceNMR were measured using a Bruker Avance 700 (700 MHz, equipped with a 5mm TCI cryoprobe, manufactured by Bruker Biospin).

Example 1 Synthesis of ¹³C/¹⁵N labeled MPC Synthesis of2-((2-cyanoethoxy) (diisopropylamino)phosphinooxy)ethyl methacrylate (ofFormula (m6))

A methacrylate represented by Formula (m6) was synthesized according toReaction Formula 3.

A 200 mL Pyrex (registered trademark) flask was charged with2-hydroxyethylmethacrylate represented by Formula (m9) (0.65 mL, 5.4mmol, manufactured by Aldrich) and dehydrated methylene chloride (DCM,manufactured by Kishida Chemical Co., Ltd.), followed by vacuumconcentration. Subsequently, 15 mL of DCM, N,N-diisopropylethylamine(DIPEA) (3.5 mL, 20 mmol, manufactured by Aldrich) and2-cyanoethyl-N,N-diisopropylchloro phosphoramidite (2.0 mL, 9.0 mmol,manufactured by Wako Pure Chemical Industries, Ltd.) were added theretoin this order with a syringe, and the resulting solution was stirredunder an Ar atmosphere at 0° C. for 1 h. After diluting the reactionsolution with DCM by approximately 20 times, saturated aqueous sodiumbicarbonate was added thereto for extraction, and the resulting solutionwas dehydrated with anhydrous MgSO₄ (manufactured by Nacalai Tesque),followed by vacuum concentration with an evaporator. The residue waspurified by silica gel column chromatography (hexane (manufactured byNacalai Tesque): ethyl acetate (manufactured by Nacalai Tesque)=4:1),and thus, the methacrylate derivative represented by Formula (m6) (acolorless viscous liquid) was quantitatively obtained.

The ¹H NMR spectrum of Formula (m6) is illustrated in FIG. 1, and thespectrum data thereof are as follows:

¹H NMR spectrum (300 MHz, CD₂Cl₂) δ/ppm=6.01-6.04 (1H, CH₂═C—),5.48-5.51 (1H, CH₂═C—), 4.21 (t, J=4.94 Hz, 2H, —CH₂OCO), 3.67-3.87 (4H,—OCH₂CH₂—OP—, —POCH₂—), 3.46-3.59 (2H, —NCH(CH₃)₃), 2.54 (t, J=6.23 Hz,2H, —CH₂CN), 1.86 (s, 3H, CH₃C═CH₂), 1.04-1.10 (12H, —NCH(CH₃)₂)

Synthesis of ¹³C/¹⁵N labeled MPC (of Formula (j1) (wherein a=2 and b=2)

A ¹³C/¹⁵N labeled MPC represented by Formula (j1) (wherein a=2 and b=2)was synthesized according to Reaction Formula 4.

A 300 mL Pyrex (registered trademark) flask was charged with a compoundrepresented by Formula (m5) (0.559 g, 3.91 mmol) and tetrazole (0.292 g,4.12 mmol, manufactured by Nacalai Tesque), followed by vacuum drying.To the resultant mixture, a dehydrated acetonitrile solution (70 mL) of50 mL of dehydrated acetonitrile (manufactured by Wako Pure ChemicalIndustries, Ltd.) and a compound represented by Formula (m6) (1.61 g,4.88 mmol) were added with a syringe, and the resulting solution wasstirred under an Ar atmosphere at room temperature for 14 h, so as toobtain a compound represented by Formula (m7) as a mixture (ESI-TOF MSm/z: 337.1652). Incidentally, the compound represented by Formula (m5)was prepared by a method described in PTL 1.

Subsequently, 60 mL of 0.1 M I₂ in THF/pyridine/water solution(manufactured by Glen Research) was added to the reaction solution, andthe resulting solution was stirred at room temperature for 2 h, so as toobtain a compound represented by Formula (m8) as a mixture (ESI-TOF MSm/z: 353.1575). After subjecting the reaction solution to vacuumconcentration, the resulting residue was subjected to two azeotropicoperations with dehydrated toluene. To the residue, 50 mL of dehydratedacetonitrile and 54 mL of a 28% aqueous ammonia solution (manufacturedby Wako Pure Chemical Industries, Ltd.) were added, followed by stirringat room temperature for 1 h. After confirming elimination of thecompound represented by Formula (m8) by ESI-TOF MS (m/z: 353.1575changed to m/z: 300.1327), the resultant mixture was subjected to vacuumconcentration with an evaporator. Ultrapure water was added to theresidue, the resultant solution was washed with dichloromethane, andthen the aqueous layer was concentrated with an evaporator and driedunder reduced pressure. After the thus-obtained crude product waspurified using alumina column chromatography(dichloromethane:methanol:water=12:6:1), methanol (manufactured byNacalai Tesque) and amberlite (IRA96SB, manufactured by OrganoCorporation) were added to the residue, and the resulting solution wasstirred for 30 min. Thereafter, the amberlite was filtered out, and theresultant solution was concentrated and dried under reduced pressure, soas to obtain a ¹³C/¹⁵N labeled MPC represented by Formula (j1) (whereina=2 and b=2) (a yellow viscous solid, 1.056 g, 3.52 mmol). The isolatedyield was 90%.

The ¹H NMR spectrum of Formula (j1) is illustrated in FIG. 2, and thespectrum data and the MS data thereof are as follows:

¹H NMR spectrum (300 MHz, D₂O) δ/ppm=6.05-6.08 (1H, CH₂═C—), 5.52-5.66(1H, CH₂═C—), 4.13-4.22 (2H, —OCH₂CH₂—OP—), 4.23-4.30 (2H,—OCH₂CH₂—OP—), 4.00-4.08 (2H, —OCH₂CH₂N), 3.50-3.58 (2H, —OCH₂CH₂N),3.09 (dt, J=144.7, 3.30 Hz, 9H, ¹⁵N(¹³CH₃)₃), 1.82 (t, J=1.28 Hz, 3H,—CH₃), ESI-TOF MS m/z: 300.1327 (M⁺+H).

The ¹³C NMR spectrum of Formula (j1) is illustrated in FIG. 3, and thespectrum data thereof are as follows:

¹³C NMR spectrum (100 MHz, D₂O/CD₃OD) δ/ppm=171.00, 137.26, 128.55,67.41 (dd, ²J_(CP)=7.85, ²J_(CN)=4.55 Hz), 65.92 (d, ²J_(CP)=7.65 Hz),65.39 (m), 60.91, 55.43 (d, ¹J_(CN)=5.27 Hz), 18.86.

The ¹⁵N NMR spectrum of Formula (j1) is illustrated in FIG. 4, and thespectrum data thereof are as follows:

¹⁵N NMR spectrum (40 MHz, D₂O/CD₃OD) δ/ppm=43.4 (q, ¹J_(CN)=5.22 Hz).

The ³¹P NMR spectrum of Formula (j1) is illustrated in FIG. 5, and thespectrum data thereof are as follows:

³¹P NMR spectrum (160 MHz, D₂O) δ/ppm=1.69.

Example 2 Synthesis of ¹³C/¹⁵N labeled PMPC (of Formula (p2)

A ¹³C/¹⁵N labeled PMPC represented by Formula (p2) was synthesizedaccording to Reaction Formula 5:

A 2 mL vial was charged with a ¹³C/¹⁵N labeled MPC represented byFormula (j1) (wherein a=2 and b=2) (135.6 mg, 0.46 mmol, 43 equiv,target number average molecular weight (M_(n))=13000) and 0.3 mL ofdehydrated methanol (manufactured by Nacalai Tesque), and Ar was allowedto flow through it for 30 min. Operations performed thereafter wereconducted in a glove compartment under a nitrogen atmosphere. Apolymerization initiator represented by Formula (p1) (3.5 mg, 0.01 mmol,1 equiv) and 0.1 mL of a dehydrated methanol solution of CuBr (1.5 mg,0.01 mmol, 1 equiv, manufactured by Wako Pure Chemical Industries, Ltd.)and 2,2′-bipyridine (3.1 mg, 0.02 mmol, 2 equiv, manufactured by NacalaiTesque) were prepared. This solution was added to the reaction solutioncontaining Formula (j1), and the resulting reaction solution was stirredat room temperature for 72 h. The solution became reddish brown. The endof the reaction was confirmed by ¹H NMR (monomer conversion of 98%).After stopping the reaction, the solution was passed through a thinsilica gel layer to remove Cu (eluent: methanol), and the eluate wasconcentrated with an evaporator. Then, methanol and dehydrated THF wereadded to the residue, and the precipitate was collected, washed withdehydrated THF, and dried under reduced pressure. The crude product waspurified by GPC (used column: SB-803HQ, eluent solvent: ultrapure water,flow rate: 1 mL/min, column temperature: 40° C.), and the purifiedproduct was lyophilized, so as to obtain a ¹³C/¹⁵N labeled PMPCrepresented by Formula (p2) (white solid, Mn=6800). The isolated yieldwas 30%. The polymer represented by Formula (p2) prepared in thisexample is sometimes abbreviated as PMPC p2-1 (having n of Formula (p2)of 23, M_(n) (GPC) of 6800 and M_(n) (NMR) of 12000), PMPC p2-2 (havingn of Formula (p2) of 16, M_(n) (GPC) of 5000 and M_(n) (NMR) of 10000),or PMPC p2-3 (having n of Formula (p2) of 40, M_(n) (GPC) of 12000 andM_(n) (NMR) of 18000).

The ¹H NMR spectrum of PMPC p2-3 is illustrated in FIG. 6, and thespectrum data thereof are as follows:

¹H NMR spectrum (400 MHz, D₂O) δ/ppm=6.54 (m, furan), 5.21 (m, furan),4.15-4.31 (br, —OCH₂CH₂OP—, —NCH₂CH₂O—), 4.04-4.15 (br, —OCH₂CH₂OP—),3.82-4.04 (br, POCH₂CH₂ ¹⁵N—, —NCH₂CH₂O—), 3.51-3.62 (br,—CH₂N(¹³CH₃)₃), 3.14 (d, ¹J_(CH)=144.8 Hz, —¹⁵N(¹³CH₃)₃), 2.82-2.99(—CHCON), 1.49-2.00 (br, —CH₂—, main chain, —C(CH₃)₂), 0.64-1.05 (br,—CH₃, main chain).

The ¹³C NMR spectrum of PMPC p2-3 is illustrated in FIG. 7, and thespectrum data thereof are as follows:

¹³C NMR spectrum (100 MHz, D₂O/CD₃OD) labeled for ¹³C δ/ppm=55.0 (d,1J_(CN)=5.02 Hz).

The ¹⁵N NMR spectrum of PMPC p2-3 is illustrated in FIG. 8, and thespectrum data thereof are as follows:

¹⁵N NMR spectrum (40 MHz, D₂O/CD₃OD) labeled for ¹⁵N δ/ppm=42.0 (q,J_(CN)=5.21 Hz).

FIG. 9 illustrates a GPC profile of PMPC p2-1. The GPC measurement wasconducted under the following conditions: used column: SB-803HQ, eluentsolvent: 0.1 M NaNO₃ aq./0.2 wt % NaN₃, flow rate: 1 mL/min, and columntemperature: 40° C. Each sample was filtered by a Millex-GV (0.22 μm)filter (Millipore Corporation) before injection.

Example 3

The equivalent amount of a monomer (of Formula (j1) (wherein a=2 andb=2)) to a synthesized polymerization initiator (of Formula (p1)) waschanged in ¹³C/¹⁵N labeled PMPC (PMPC p2-2 and PMPC p2-3) havingdifferent molecular weights, and an operation similar to that of Example2 was performed, so as to obtain PMPC p2-2 having M_(n) calculated basedon the GPC of 5000 and a PMPC p2-3 having M_(n) of 12000. The GPC chartsof these PMPCs are illustrated in FIGS. 10 and 11, respectively. Inaddition, data of PMPC p2-1 to p2-3 are listed together in Table 1below.

TABLE 1 ¹³C/¹⁵N Monomer labeled Monomer Target conversion M_(n) M_(n)Yield PMPC unit M_(n) (%) (NMR) (GPC) ^(a)) PDI (%) ^(b)) PMPC 43 1300098 12000 6800 1.17 30 p2-1 PMPC 35 10000 94 10000 5000 1.1 3 p2-2 PMPC71 20000 94 18000 12000 1.14 13 p2-3 ^(a)) PEG standard ^(b)) Isolatedyield based on GPC

In 72 h, a ¹³C/¹⁵N labeled PMPC was obtained with monomer conversion of94 to 98% with an isolated yield of 3 to 30%. As a result of GPCmeasurement of molecular weight calibrated with poly(ethylene glycol)(PEG), polydispersity index (PDI) was found to have a value of 1.10 to1.17, which shows that the synthesized ¹³C/¹⁵N labeled PMPC polymer wasquite close to being monodisperse.

Furthermore, when no polymerization initiator was added to the reactionsolution as a reference experiment, the polymerization reaction did notproceed at all, and the targeted polymer could not be obtained (asconfirmed by ¹H NMR and GPC). This result suggests that spontaneouspolymerization of the ¹³C/¹⁵N labeled MPC did not occur but a livingradical polymerization reaction had proceeded from the polymerizationinitiator.

Example 4 Setting of INEPT-Based Pulse Sequence for Triple Resonance NMR

An INEPT-based pulse sequence for triple resonance illustrated in FIG.12 was used. In FIG. 12, the narrow and broad filled bars represent 90°pulses and 180° pulses, respectively.

Based on the ¹H NMR of the ¹³C/¹⁵N labeled PMPC p2-3, peaks attributedto ¹H derived from a furan ring of the polymerization initiator wereobserved at 6.54 ppm and 5.21 ppm. In addition, a doublet peak of amethyl group coupled with ¹³C was observed, and a coupling constant(¹J_(CH)) of ¹H—¹³C was calculated as ¹J_(CH)=144.8 Hz (FIG. 6).Furthermore, a peak of a methyl group at 55.0 ppm was confirmed in the¹³C NMR, a peak of quaternary ammonium at 42.0 ppm was confirmed in the¹⁵N NMR, and a coupling constant (¹J_(CN)) of ¹³C—¹⁵N was calculated as¹J_(CN)=5.21 Hz (FIGS. 7 and 8). Based on these data, a ¹H—{¹³C—¹⁵N}triple resonance signal of the PMPC p2-1 can be selectively observed bysetting offsets (chemical shifts) of ¹³C and ¹⁵N respectively to¹³C=55.0 ppm and ¹⁵N=42.0 ppm and setting delay intervals (couplingconstants) of FIG. 12 to ¼¹J_(CH)=1.73 ms and ¼¹J_(CN)=48 ms.

Example 5 ¹H—{¹³C—¹⁵N} Triple Resonance NMR of ¹³C/¹⁵N Labeled PMPC p2-3in a Complex System

The NMR spectrum of PMPC p2-3 was measured in a mouse liver extractincluding a large number of proteins and amino acids, so as to examinethe influence of the molecular weight increase on the selectivity for a¹H-{¹³C—¹⁵N} triple resonance NMR signal.

First, mouse liver tissues were harvested and homogenized. Thehomogenate and the PMPC p2-3 in 1 μM (a molar concentration calculatedusing M_(n) (GPC)) were mixed and lyophilized to dryness, dissolved in aD₂O solution, and subjected to NMR analysis.

In the general ¹H NMR, a large number of ¹H signals were observed, andhence it was impossible to detect a ¹H signal derived from a methylgroup of the PMPC p2-3 (FIG. 13A).

In contrast, in a ¹H—{¹³C} double resonance NMR spectrum of the samesample, although the selectivity was improved as compared with that inthe ¹H NMR, ¹H signals derived from contaminations included in the liverextract were so strongly observed that it was impossible to detecthighly selectively a ¹H signal derived from a methyl group of PMPC p2-3(FIG. 13B). When the measurement was further performed using the¹H—{¹³C—¹⁵N} triple resonance NMR, only a ¹H signal at 3.14 ppm derivedfrom a methyl group of the PMPC p2-3 was observed (FIG. 13C).

It was shown, based on the aforementioned results, that a ¹H signalderived from a methyl group of PMPC p2-3 can be highly selectivelydetected even in an organism system containing a large number ofcontaminations by employing ¹H—{¹³C—¹⁵N} triple resonance NMR.

In the ¹H—{C—¹⁵N} triple resonance NMR spectrum, a noise peak wasobserved at 3.67 ppm (FIG. 13C). For this peak, there is a possibilitythat noise derived from magnetization remaining in a sequence, otherthan the ¹H—¹³C—¹⁵N sequence, included in the contaminations isobserved. However, it is considered that this noise intensity may belowered by optimizing a parameter of the phase cycling or the like ofthe pulse sequence. With respect to the ¹³C/¹⁵N labeled PMPC p2-3 (1 μM(a molar concentration calculated using M_(n) (GPC)) in a 10 mM tris-HClbuffer (pH 8.0) including a mouse liver extract (10% v/v) and2-mercaptoethanol (0.5 mM), the ¹H NMR (FIG. 13A), the ¹H—{¹³C} doubleresonance NMR (FIG. 13B) and the ¹H—{¹³C—¹⁵N} triple resonance NMR(number of scans: 256) (FIG. 13C) are shown.

Example 6 Macromolecular Effect (Effect of Accumulation of StableIsotopes) on Signal Sensitivity

Next, the macromolecular effect on the signal sensitivity was examined.Considering application to MR imaging, three types of ¹³C/¹⁵N labeledPMPC (p2-1 to p2-3) having M_(n) (GPC) of 5000 (16 units), 6800 (23units) and 12000 (40 units) were evaluated in D₂O with a measuring timeof 8 min (number of scans: 256) by using the ¹H—{¹³C—¹⁵N} tripleresonance NMR (FIG. 14A). In all of the ¹³C/¹⁵N labeled PMPC having anymolecular weight, a signal-noise ratio (S/N) was linearly decreasedalong with a decrease in the polymer concentration. Furthermore, whenthe concentrations were the same, the S/N was increased in proportion tothe number of monomer units. The ¹³C/¹⁵N labeled MPC (of Formula (j1))(wherein a=2 and b=2) of 1 μM had a S/N of 8.72, but the ¹³C/¹⁵N labeledPMPC p2-3 of 1 μM (a molar concentration calculated by M_(n) (GPC))having stable isotopes accumulated by increasing the molecular weightattained S/N of 221, and thus, the signal intensity was increased byapproximately 25 times. Furthermore, the detection limit of the ¹³C/¹⁵Nlabeled PMPC p2-3 was checked, and the ¹³C/¹⁵N labeled PMPC even at 50nM (corresponding to 30 nM calculated as a molar concentration by usingM_(n) (NMR)) had a S/N of 17.5, and a clear signal was observed (FIG.14B).

It was shown, based on these results, that the signal intensity can beremarkably increased so as to be detectable with high sensitivity of theorder of nM in ¹³C/¹⁵N labeled PMPC in which stable isotopes areaccumulated by increasing the molecular weight. A graph illustrating therelationship between the concentration (molar concentration calculatedusing the M_(n) (GPC)) and the S/N of the ¹³C/¹⁵N labeled MPC and the¹³C/¹⁵N labeled PMPC p2-1 to p2-3 obtained by the ¹H—{¹³C—¹⁵N} tripleresonance NMR performed in D₂O is given in FIG. 14A, and ¹H—{¹³C—¹⁵N}triple resonance NMR spectra (number of scans: 256) in D₂O obtained atvarious concentrations (from 50 nM to 1 μM: a molar concentrationcalculated by M_(n) (GPC)) of the ¹³C/¹⁵N labeled PMPC p2-3 are given inFIG. 14B.

Example 7 Preparation of Single Chain Antibody hu4D5-8scFv

Based on a gene sequence (hu4D5-8) of a variable region of IgG bound toHer2, a gene hu4D5-8scFv encoding a single chain antibody (scFv) wasproduced. First, VL and VH genes of the hu4D5-8 were linked by a cDNAencoding peptide (GGGGS) 3 so as to produce a cDNA. A restriction enzymeNcoI- was introduced into the 5′-end, and a recognition site for arestriction enzyme NotI was introduced into the 3′-end. The nucleotidesequence is as follows:

(Seq. ID Number 1) 5′-CCATGGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGATCCAGATCTGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTACTCCTCCCACGTTCGGACAGGGTACCAAGGTGGAGATCAAAGGCGGTGGTGGCAGCGGTGGCGGTGGCAGCGGCGGTGGCGGTAGCGAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATTAAAGACACCTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAAGGATTTATCCTACGAATGGTTATACTAGATATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTGCAGATGAACAGCCTGCGTGCTGAGGACACTGCCGTCTATTATTGTTCTAGATGGGGAGGGGACGGCTTCTATGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCGGCCGC-3′

(The recognition sites for the restriction enzymes are underlined.)

This gene fragment hu4D5-8scFv was inserted downstream of the T7/lacpromoter of plasmid pET-22b (+) (manufactured by Novagen, Inc.).Specifically, the cDNA obtained above was ligated to pET-22b (+) havingbeen subjected to a digestion treatment with the restriction enzymesNcoI- and NotI.

The resulting expression plasmid was transformed into Escherichia coliBL21 (DE3) so as to obtain a strain for expression. The thus obtainedstrain was cultured in 4 mL of an LB-Amp medium overnight, and the wholevolume was added to 250 mL of a 2xYT medium, followed by shaking theculture at 28° C. and 120 rpm for 8 hours. Thereafter, IPTG(isopropyl-β-D (−)-thiogalactopyranoside) was added thereto to attain afinal concentration of 1 mM, and the resultant mixture was culturedovernight at 28° C. The cultured Escherichia coli were subjected tocentrifugal separation at 8000×g and 4° C. for 30 min, and thesupernatant culture broth was collected. Ammonium sulfate in an amountcorresponding to 60 wt % of the culture broth was added to thethus-obtained culture broth to precipitate the protein by salting out.The solution having been subjected to the salting out was allowed tostand at 4° C. overnight and then subjected to centrifugal separation at8000×g and 4° C. for 30 min so as to collect the precipitate. Theobtained precipitate was dissolved in a 20 mM tris HCl/500 mM NaClbuffer and dialyzed with 1 L of this buffer. The dialyzed proteinsolution was added to a column filled with H is Bind (registeredtrademark) Resin (manufactured by Novagen, Inc.) and purified by metalchelate affinity chromatography using Ni ions. The thus purifiedhu4D5-8scFv was confirmed, by reduction SDS-PAGE, to appear as a singleband and to have a molecular weight of approximately 27 kDa. The aminoacid sequence of the prepared antibody is as follows. Hereinafter,hu4D5-8scFv is abbreviated as scFv.

(Seq. ID Number 2) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAAALEHHH HHHGGC

Example 8

The ¹³C/¹⁵N labeled PMPC p2-1 (of Formula (p2)) was used as a polymertag that transmits a multiple resonance signal so as to tag scFv thatwas an artificial antibody having a region for recognizing a Her2antigen of breast cancer (Reaction Formula 6).

(Retro-Diels-Alder Reaction of Protecting Group at an End of thePolymer)

A glass reaction tube was charged with the ¹³C/¹⁵N labeled PMPC p2-1(M_(n) (GPC)=6800, M_(n) (NMR)=12000) (34.8 mg, 4.99 mmol) obtained inExample 2 and 1 mL of dehydrated toluene (manufactured by Wako PureChemical Industries, Ltd.), followed by heating at reflux under an Aratmosphere for 48 h. After stopping the reaction, the resultant solutionwas concentrated with an evaporator and dried under reduced pressure. Tothe residue, methanol and dehydrated THF were added, and a precipitatewas collected to obtain a PMPC p3 (yellow solid) represented by Formula(p3). The ¹H NMR measurement was conducted on the obtained polymer,resulting in the finding that a peak derived from a furan ring had beeneliminated, and a signal derived from a maleimide group was observed at6.85 ppm.

(Immobilization of scFv onto ¹³C/¹⁵N Labeled PMPC)

Next, a 1.5 mL Snap Lock Micro-tube was charged with 100 μL of 7.75 μMscFv (5 mM PBS-EDTA buffer) and 1.5 μL of 10 mMtris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) (5 mM PBS-EDTAbuffer), and the resulting solution was slowly mixed at room temperaturefor 2 h. Subsequently, 1.47 μL of an aqueous solution of the PMPC p3 (10mM) filtered by a Millex-LH (0.45 μm) filter (Millipore Corporation) wasadded thereto, and the resulting solution was slowly mixed at roomtemperature for 4 h. Thereafter, 1.5 μL of 10 mM L-cysteine(manufactured by Nacalai Tesque) (5 mM PBS-EDTA buffer) was addedthereto, and the resulting solution was slowly mixed at room temperaturefor 30 min.

The reaction solution was transferred to an Amicon Ultra filtration tube(molecular weight cutoff 10 KDa) and concentrated by centrifuging(14000×g) for 20 minutes. To the concentrated solution, 100 μL of abinding buffer (1× phosphate, 20 mM imidazole) was added, and theresultant was centrifuged (14000×g) for 10 min. This operation wasrepeated twice with substitution of the buffer. The mixture was purifiedusing a Ni affinity column (H is Spin Trap (trademark), GE Healthcare)and concentrated using an Amicon Ultra filtration tube (molecular weightcut off 10 KDa), and to the resultant solution, 400 μL of 5 mM PBS-EDTAwas added, followed by centrifuging (14000×g) for 30 min. This operationwas repeated twice, so as to obtain a mixture of PMPC p4 (of Formula(p4))-scFv conjugate and unreacted scFv. Incidentally, production of thePMPC p4-scFv conjugate was confirmed using an SDS-PAGE (4-20%)measurement.

Example 9 ¹H—{¹³C—¹⁵N} Triple Resonance NMR of ¹³C/¹⁵N Labeled PMPC p4Having scFv Immobilized Thereon

The NMR spectrum of the ¹³C/¹⁵N labeled PMPC p4 (of Formula (p4))-scFvconjugate was measured. In the general ¹H NMR, a ¹H signal derived froma methyl group of the ¹³C/¹⁵N labeled PMPC as well as all ¹H signalsderived from scFv and the buffers were observed (FIG. 15A). In the¹H—{¹³C} double resonance NMR, a ¹H signal derived from a methyl groupof the ¹³C/¹⁵N labeled PMPC-scFv conjugate was more clearly detected at3.14 ppm, but a ¹H signal derived from scFv or the buffer was alsoobserved at 3.55 ppm (FIG. 15B).

In contrast, when the ¹H—{³C—¹⁵N} triple resonance NMR was measured,only a ¹H signal derived from a methyl group of the ¹³C/¹⁵N labeledPMPC-scFv conjugate was observed (FIG. 15C). It was confirmed, based onthese results, that an anti-Her2 artificial antibody scFv can be taggedwith a ¹³C/¹⁵N labeled PMPC and can be highly selectively detected by¹H—{¹³C—¹⁵N} triple resonance NMR. Consequently, it was shown that apolymer having, in its side chains, the ¹H—¹³C—¹⁵N sequence representedby Formula (y1) can be highly selectively detected, and according to asimilar principle, the ¹H—¹³C—¹³C sequence represented by Formula (y2)and the ¹H—¹⁵N—¹³C sequence represented by Formula (y3) can be similarlyhighly selectively detected.

With respect to the ¹³C/¹⁵N labeled PMPC p4-scFv conjugate, the ¹H NMR(FIG. 15A), the ¹H—{¹³C} double resonance NMR (FIG. 15B) and the¹H—{¹³C—¹⁵N} triple resonance NMR (700 MHz, in D₂O) (FIG. 15C) areshown.

While the present invention has been described with reference toexemplary embodiments, it is to be understood that the invention is notlimited to the disclosed exemplary embodiments. The scope of thefollowing claims is to be accorded the broadest interpretation so as toencompass all such modifications and equivalent structures andfunctions.

This application claims the benefit of Japanese Patent Application No.2012-118061, filed May 23, 2012, which is hereby incorporated byreference herein in its entirety.

1. A polymer comprising, in a main chain, one or a plurality ofrepeating unit(s) selected from the group consisting of formulas (x1) to(x3), the polymer having a degree of polymerization of two or more and5000 or less, wherein a side chain of each of the repeating unit(s) hasa structure selected from the group consisting of formulas (y1) to (y3):

wherein: in the formulas (x1) to (x3), each of R¹ to R⁴ independentlyrepresents a hydrogen atom, or a substituted or unsubstitutedhydrocarbon group having one or more and four or less carbon atoms; R⁵represents a substituted or unsubstituted hydrocarbon group having oneor more and nine or less carbon atoms, and if R⁵ is a hydrocarbon grouphaving two or more carbon atoms, any of the carbon atoms may be bound tothe side chain, in the formulas (x1) to (x3), * (an asterisk) representsa bond to the side chain directly or via a linker, in the formulas (y1)to (y3), * (an asterisk) represents a bond to the main chain directly orvia a linker, in the formulas (y1) to (y3), Z represents a monovalentatom or a monovalent atom group, in the formula (y3), R⁶ represents adirect bond, or a substituted or unsubstituted hydrocarbon group havingone or more and four or less carbon atoms, and a substituent of each ofR¹ to R⁶ is a functional group including at least one selected from thegroup consisting of a halogen atom, an oxygen atom, and a nitrogen atom.2. The polymer according to claim 1, wherein the linker and the sidechain are selected from the group consisting of formulas (y5) to (y7):

wherein: in the formulas (y5) to (y7), * (an asterisk) represents a bondto the main chain, each of a and b independently represents an integerof one or more and four or less, and a hydrogen atom of a methylenegroup in the formulas (y5) to (y7) is optionally replaced by anotheratom.
 3. The polymer according to claim 1, wherein a structurecomprising the repeating unit(s) is represented by formula (I):

wherein: in the formula (I), X represents any one of the formulas (x1)to (x3), the polymer has a degree of polymerization of two or more and5000 or less, L represents a direct bond, or a divalent atom or adivalent atom group, if L is the divalent atom or the divalent atomgroup, L binds to X or Y of the formula (I), and Y is represented by anyone of the formulas (y1) to (y3):

in the formulas (x1) to (x3), * (an asterisk) represents a bond to L ofthe formula (I), or a bond to Y if L is a direct bond, each of R¹ to R⁴independently represents a hydrogen atom, or a substituted orunsubstituted hydrocarbon group having one or more and four or lesscarbon atoms, R⁵ represents a substituted or unsubstituted hydrocarbongroup having one or more and nine or less carbon atoms, and asubstituent of each of R¹ to R⁵ is a functional group including at leastone selected from the group consisting of a halogen atom, an oxygenatom, and a nitrogen atom,

in the formulas (y1) to (y3), * (an asterisk) represents a bond to L ofthe formula (I), or a bond to X if L is a direct bond, Z represents amonovalent atom or a monovalent atom group, R⁶ of the formula (y3)represents a direct bond, or a substituted or unsubstituted hydrocarbongroup having one or more and four or less carbon atoms, and asubstituent of R⁶ is a functional group including at least one selectedfrom the group consisting of a halogen atom, an oxygen atom, and anitrogen atom.
 4. The polymer according to claim 3, wherein L of theformula (I) is selected from the group consisting of a substituted orunsubstituted hydrocarbon group having one or more and four or lesscarbon atoms and formulas (l1) to (l3):

wherein: in the formulas (l1) to (l3), * (an asterisk) represents a bondto X or Y of the formula (I), a substituent of the hydrocarbon group isa functional group including at least one selected from the groupconsisting of a halogen atom, an oxygen atom, and a nitrogen atom, andL′ of the formulas (l1) to (l3) is selected from the group consisting ofa substituted or unsubstituted hydrocarbon group having one or more andfour or less carbon atoms and formula (l′):

wherein: in the formula (l′), each of a and b independently representsan integer of one or more and four or less, and the hydrocarbon grouphaving one or more and four or less carbon atoms of L′ and a hydrogenatom of a methylene group of the formula (l′) are optionally replaced byanother atom.
 5. The polymer according to claim 1, wherein Z of theformula (y1) or (y2) is represented by formula (z1) or Z of the formulas(y1) to (y3) is represented by (z2):*—¹⁵NH₂  (z1)*—¹³CH₃  (z2) wherein: in the formulas (z1) and (z2), *(an asterisk)represents a bond to ¹³C or ¹⁵N of the formulas (y1) to (y3).
 6. Thepolymer according to claim 1, wherein Z of the formulas (y1) to (y3) isrepresented by formulas (z3) or (z4):*—(CH₂)_(d)—COO⁻  (z3)*—(CH₂)_(d)—SO₃ ⁻  (z4) wherein: in the formulas (z3) and (z4), * (anasterisk) represents a bond to ¹³C or ¹⁵N of the formulas (y1) to (y3),and in the formulas (z3) and (z4), d represents an integer of one ormore and four or less, and a hydrogen atom of a methylene group isoptionally replaced by another atom.
 7. The polymer according to claim1, represented by any one of formulas (i1) to (i12):

wherein, in the formulas (i1) to (i12), each of a and b independentlyrepresents an integer of one or more and four or less, and a hydrogenatom of a methylene group is optionally replaced by another atom.
 8. Thepolymer according to claim 1, wherein the polymer has a degree ofpolymerization of 10 or more and 400 or less.
 9. The polymer accordingto claim 1, wherein the polymer has, at an end of the repeating unit(s),any one of an N-hydroxysuccinimide group, a maleimide group, an aminogroup, an azide group, an ethynyl group, a vinyl group, a trichlorosilylgroup, a thiol group, a hydroxyl group, and an alkyl group.
 10. Acompound comprising the polymer according to claim 1 and a trappingmolecule that specifically binds to a target site.
 11. The polymeraccording to claim 1, comprising two or more repeating units.
 12. Thepolymer according to claim 1, comprising merely one repeating unit. 13.The polymer according to claim 1, comprising a repeating unit derivedfrom a methacrylate monomer, a repeating unit derived from amethacrylamide monomer, a repeating unit derived from an amino acidmonomer, and a repeating unit derived from a hydroxy acid monomer. 14.The polymer according to claim 1, comprising a repeating unitrepresented by any one of formulas (a1) to (a3):

wherein, in the formulas (a1) to (a3), R⁷ represents a hydrogen atom, ora substituted or unsubstituted hydrocarbon group having one or more andsix or less carbon atoms, and a substituent of R⁷ is a functional groupincluding at least one selected from the group consisting of a halogenatom, an oxygen atom, and a nitrogen atom.
 15. The polymer according toclaim 11, wherein the polymer is an alternating copolymer, a randomcopolymer or a block copolymer.
 16. A contrast agent for nuclearmagnetic resonance analysis or magnetic resonance imaging, comprisingthe polymer according to claim 1 and a dispersion medium.
 17. A compoundrepresented by any one of formulas (j1) to (j12):

wherein, in the formulas (j1) to (j12), each of a and b independentlyrepresents an integer of one or more and four or less, and a hydrogenatom of a methylene group is optionally replaced by another atom.
 18. Amethod of nuclear magnetic resonance analysis, comprising detecting apolymer in a specimen, the method comprising: preparing the polymeraccording to claim 1; providing the polymer to the specimen; andapplying electromagnetic waves to the specimen provided with thepolymer, wherein magnetization transfer (coherence transfer) amongnuclei in a ¹H—¹³C—¹⁵N bond sequence, a ¹H—¹⁵N—¹³C bond sequence or a¹H—¹³C—¹³C bond sequence of the polymer is utilized for detecting thepolymer.
 19. A method of magnetic resonance imaging, comprisingdetecting a position of a polymer in a specimen, the method comprising:preparing the polymer according to claim 1; providing the polymer to thespecimen; and applying electromagnetic waves to the specimen providedwith the polymer, wherein magnetization transfer (coherence transfer)among nuclei in a ¹H—¹³C—¹⁵N bond sequence, a ¹H—¹⁵N—¹³C bond sequenceor a ¹H—¹³C—¹³C bond sequence of the polymer is utilized for detectingthe position of the polymer.
 20. The method according to claim 18,wherein the magnetization transfer (coherence transfer) among nuclei inthe ¹H—¹³C—¹⁵N bond sequence is utilized.
 21. A contrast agent fornuclear magnetic resonance analysis or magnetic resonance imaging,comprising: a polymer, wherein the polymer comprises, in a main chain,one or a plurality of repeating unit(s) selected from the groupconsisting of formulas (x1) to (x3), and the polymer has, in a sidechain bound to the main chain directly or via a linker in a positioncorresponding * (an asterisk) of the formulas (x1) to (x3), a structureselected from the group consisting of a ¹H—¹³C—¹⁵N bond sequence, a¹H—¹⁵N—¹³C bond sequence, and a ¹H—¹³C—¹³C bond sequence:

wherein: in the formulas (x1) to (x3), each of R¹ to R⁴ independentlyrepresents a hydrogen atom, or a substituted or unsubstitutedhydrocarbon group having one or more and nine or less carbon atoms, R⁵represents a substituted or unsubstituted hydrocarbon group having oneor more and four or less carbon atoms, and a substituent of R⁵ is afunctional group comprising at least one selected from the groupconsisting of a halogen atom, an oxygen atom, and a nitrogen atom.